FABP5 deletion in nociceptors augments endocannabinoid signaling and suppresses TRPV1 sensitization and inflammatory pain

The endocannabinoid anandamide (AEA) produces antinociceptive effects by activating cannabinoid receptor 1 (CB1). However, AEA also serves as an agonist at transient receptor potential vanilloid receptor 1 (TRPV1) in nociceptive sensory neurons, which may exacerbate pain. This potential functional duality is highlighted by the failure of an inhibitor of the AEA catabolic enzyme fatty acid amide hydrolase (FAAH) to afford pain relief in a clinical trial. Consequently, it remains to be determined whether elevating AEA levels in nociceptors leads to antinociceptive or pro-nociceptive effects. Fatty acid binding protein 5 (FABP5) is an intracellular carrier that mediates AEA transport to FAAH for inactivation. Leveraging the abundant expression of FABP5 in TRPV1+ nociceptors, we employed a conditional knockout strategy to demonstrate that FABP5 deletion in nociceptors augments AEA levels, resulting in the emergence of antinociceptive effects mediated by CB1. Mechanistically, FABP5 deletion suppresses inflammation- and nerve growth factor-mediated TRPV1 sensitization via CB1, an effect mediated by calcineurin. Unexpectedly, inhibition of FAAH failed to blunt TRPV1 sensitization, uncovering functionally distinct outputs resulting from FABP5 and FAAH inhibition. Collectively, our results demonstrate that FABP5 serves a key role in governing endocannabinoid signaling in nociceptors to disrupt TRPV1 sensitization and pain, and position FABP5 as a therapeutic target for the development of analgesics.

It is well-established that enhancement of peripheral AEA signaling via inhibition of its catabolic enzyme fatty acid amide hydrolase (FAAH) produces antinociceptive effects mediated by CB1 and/or CB2 receptors 18,19 . However, a clinical trial of a FAAH inhibitor failed to demonstrate clinically meaningful analgesia 20 . A plausible mechanism to explain this discrepancy stems from previous observations that FAAH substrates including AEA engage additional molecular targets that may counteract the antinociceptive effects resulting from CB1 activation. In addition to serving as a CB1 ligand, AEA is also a TRPV1 agonist and inflammatory mediators such as

Results
Inflammatory hyperalgesia in FABP5 cKO mice. FABP5 Flox mice were designed with loxP sites flanking exons 2 and 3 of the FABP5 gene (Fig. 1A,B). The mice were born at expected Mendelian frequencies and were otherwise normal. To ablate FABP5 in TRPV1 + nociceptors, we crossed FABP5 Flox mice with TRPV1-Cre mice to generate FABP5 Flox/TRPV1-Cre mice (hereafter referred to as FABP5 cKO). Reduced FABP5 expression in DRGs of FABP5 cKO mice was confirmed via qPCR and histologically (Fig. 1C,D, Supplementary Fig. 1). Note that residual FABP5 immunostaining in FABP5 cKO DRGs was observed in cells surrounding sensory neurons (Fig. 1D, arrowheads), presumably reflecting FABP5 expression in satellite glial cells 46 .
We previously reported that pharmacological or genetic inhibition of FABP5 produces antinociceptive effects in models of inflammatory pain [43][44][45]48,49 . Consequently, we assessed inflammatory hyperalgesia in mice receiving an intraplantar injection of carrageenan 49 . As expected, WT and FABP5 Flox mice developed thermal hyperalgesia while paw withdrawal latencies were significantly higher in global FABP5 KO and FABP5 cKO mice ( Fig. 2A) 50 , providing evidence that FABP5 deletion in sensory neurons produces antinociceptive effects. Comparable effects were observed in female mice (Fig. 2B), indicating that the function of FABP5 in pain modulation extends to both sexes. We additionally employed the behavioral spectrometer as a non-reflexive readout of inflammatory pain. The behavioral spectrometer quantifies diverse behavioral patterns in mice, a subset of which are pain-related and can be rescued by analgesics 51 . Of the behavioral patterns assessed, we found that carrageenan administration to male and female WT mice resulted in an increase in "still" behavior while the remaining parameters were unchanged (Fig. 2B, Supplementary Fig. 2), suggesting that mice remained more stationary in the setting of acute inflammatory pain. Administration of naproxen restored "still" patterns to pre-carrageenan baselines, confirming that these effects were pain-specific (Fig. 2B). Notably, while carrageenan administration increased "still" behavior in male and female FABP5 Flox mice, it failed to do so in FABP5 KO and FABP5 cKO mice (Fig. 2c).
Contribution of CB1 and PPARα to pain hypersensitivity in FABP5 cKO mice. To characterize the mechanisms underlying pain modulation by FABP5, we first quantified the levels of AEA, 2-AG, PEA, and OEA in DRGs of FABP5 Flox and FABP5 cKO mice. Compared to FABP5 Flox mice, AEA, PEA, and OEA levels were elevated in DRGs obtained from FABP5 cKO mice while 2-AG levels remained unchanged, and no sexspecific differences were noted (Fig. 3A). Carrageenan administration did not alter AEA, PEA, or OEA levels in FABP5 cKO mice (Fig. 3A). The expression of FAAH and the N-acylethanolamine biosynthetic enzyme NAPE-PLD were comparable between the genotypes and sexes (Fig. 3B). To validate these results, we employed the FAAH substrate AMC-AEA ( Supplementary Fig. 3) to assess FAAH activity in DRGs of both genotypes, which revealed comparable activity levels (Fig. 3C). Similarly, we observed comparable hydrolysis of the phospholipase A1 substrate PED-A1 in DRGs of these mice (Fig. 3C). Since PED-A1 is not specific for NAPE-PLD, we treated DRGs with the selective NAPE-PLD inhibitor LEI-401 to identify the enzymatic activity corresponding to NAPE-PLD ( Supplementary Fig. 3). Our results revealed comparable PED-A1 hydrolysis in DRGs of both genotypes treated with vehicle or LEI-401 (Fig. 3C). Taken together, these results indicate that the elevations in N-acylethanolamine levels observed in FABP5 cKO mice stem from attenuated transport and metabolism rather than alterations in biosynthetic or catabolic enzymes. Since no sex-specific differences were observed in any of the parameters assessed, we employed mixed cohorts of male and female mice for all subsequent experiments.

FABP5 inhibition reduces inflammation-and TRPV1-evoked CGRP release. Inflammatory
hyperalgesia is characterized by exacerbated release of CGRP from sensory neuron terminals into the dorsal horn of the spinal cord 29,30,52 . CGRP levels are elevated in rodents and humans experiencing ongoing pain and are reduced by analgesics 29,30 , thereby serving as a neurochemical proxy for nociceptor sensitization/output. We previously reported that the carrageenan-induced increase in spinal CGRP levels is blunted following global FABP5 deletion 49 . Consistent with these results, CGRP release was suppressed in both FABP5 KO and cKO mice following carrageenan challenge (Fig. 4A), confirming that FABP5 deletion in nociceptors recapitulates the effects observed in global FABP5 KO mice. TRPV1 is extensively co-expressed with CGRP and FABP5 in sensory neurons 45,53,54 and carrageenan administration sensitizes TRPV1, leading to exacerbated CGRP release 49 . Consistent with this notion, while capsaicinevoked CGRP release was enhanced in WT mice, its release was blunted in spinal cords of FABP5 KO and FABP5 cKO mice (Fig. 4B), indicating that FABP5 deletion in sensory neurons leads to attenuated TRPV1 sensitization. Administration of AM251 or GW6471 to FABP5 KO or cKO mice restored CGRP release to levels comparable to WT mice (Fig. 4B), confirming that CB1 and PPARα activation underlies these effects.

FABP5 deletion blunts TRPV1 sensitization via CB1 and PPARα. Carrageenan-evoked hyperalgesia
is dependent upon nerve growth factor (NGF) release, which plays an obligate role in TRPV1 sensitization [55][56][57][58][59] . NGF activates the TrkA receptor that is co-expressed with TRPV1 in numerous sensory neurons 60 . We confirmed that DRG neurons co-express FABP5 and TrkA (Fig. 5A) and subsequently employed calcium imaging to determine the impact of FABP5 deletion upon TRPV1 sensitization by NGF. TRPV1 undergoes rapid tachyphylaxis following repeated capsaicin application 61 that can be largely restored by mediators that sensitize TRPV1 www.nature.com/scientificreports/ including NGF (Fig. 6A). Our results revealed that while NGF sensitized TRPV1 in WT DRGs, it failed to do so in DRGs obtained from FABP5 KO mice (Fig. 6B). Comparable TRPV1 and TrkA expression was observed between the genotypes (Fig. 6C), confirming that these results do not stem from altered TRPV1 or TrkA expression. Application of the CB1 antagonist AM251 restored the ability of NGF to sensitize TRPV1 in FABP5 KO DRGs ( Fig. 6B) and is consistent with the extensive co-expression between FABP5 and CB1 (Fig. 5B). The PPARα antagonist GW6471 was likewise effective in restoring the ability of NGF to sensitize TRPV1 (Fig. 6B). CB1 and PPARα expression was comparable between the genotypes (Fig. 6C). Mechanistically, exogenously applied CB1 agonists suppress TRPV1 sensitization via calcineurin 62 . Consistent with these observations, the calcineurin inhibitor cyclosporin A largely restored TRPV1 sensitization in FABP5 KO DRGs (Fig. 6B), indicating that a common mechanism is shared between exogenous and endogenous CB1 agonists. Collectively, these data provide evidence that FABP5 deletion promotes CB1 and PPARα activation to blunt NGF-mediated TRPV1 sensitization.
To determine whether these effects are observed in vivo, we assessed capsaicin-evoked nocifensive (paw licking/biting) behaviors in WT and FABP5 KO mice pre-treated with vehicle or NGF. Intraplantar capsaicin induced comparable nocifensive behaviors in WT and FABP5 KO mice at baseline (Fig. 7). In contrast, while administration of NGF potentiated capsaicin-evoked nocifensive behaviors in WT mice, it failed to do so in mice lacking FABP5 (Fig. 7). Administration of vehicle or NGF alone did not elicit a robust behavioral response. Collectively, our results provide direct evidence that FABP5 is essential for NGF-mediated TRPV1 sensitization in vivo.

FAAH inhibition produces distinct effects upon TRPV1.
Based on the role of FABP5 as an intracellular carrier that delivers AEA to FAAH 39 , inhibition of FAAH is expected to phenocopy the effects of FABP5 upon TRPV1 sensitization. However, FAAH inhibition was previously reported to enhance TRPV1 activation in DRG neurons while intraplantar capsaicin administration exacerbated nocifensive behaviors in FAAH KO mice 7,27 . These results suggest that blockade of FAAH and FABP5 may produce non-overlapping and possibly opposing effects upon TRPV1 sensitization. We explored this possibility and found that application of the selective FAAH inhibitor PF3845 failed to blunt NGF-mediated TRPV1 sensitization despite elevating AEA levels in DRGs (Fig. 6B,D). Moreover, administration of the peripherally-restricted FAAH inhibitor URB937 (1 mg/kg, i.p.) 19 did not suppress capsaicin-evoked CGRP release (Fig. 6E,F). Taken together, these results provide further evidence that while inhibition of both FABP5 and FAAH results in elevated AEA levels, distinct effects upon TRPV1 sensitization are observed.

Discussion
Augmentation of endocannabinoid signaling represents an attractive strategy to treat pain. Initial translational efforts to harness the therapeutic potential of the endocannabinoid system were dampened following the failure of a FAAH inhibitor in a clinical trial of osteoarthritis pain 20 . A mechanism(s) underlying this seeming lack of efficacy has yet to emerge. While systemic elevations in AEA levels produce antinociceptive effects in most  www.nature.com/scientificreports/ preclinical models of pain, inflammatory mediators can convert AEA into a potent TRPV1 agonist in nociceptive sensory neurons while TRPV1-mediated nocifensive responses are observed in mice lacking FAAH 24,27 . Therefore, it is conceivable that the dual function of AEA as a CB1 and TRPV1 agonist may contribute at least in part to the observed lack of efficacy of the FAAH inhibitor. However, the molecular targets engaged by AEA in nociceptors in vivo have not been established and the goal of the current work was to determine whether FABP5 deletion and ensuing AEA elevation leads to CB1-mediated antinociceptive effects or TRPV1-mediated pro-nociceptive effects. Previous efforts to characterize the role of the peripheral endocannabinoid system in pain modulation have yielded important insights at the behavioral level. Administration of peripherally-restricted FAAH inhibitors resulted in CB1-and/or PPARα-mediated antinociceptive effects 19 , providing strong evidence that enhancement of the peripheral AEA and PEA tone is sufficient to modulate pain. Moreover, deletion of CB1 receptors from nociceptors abolished the antinociceptive effects of exogenous CB1 agonists 4 , further positioning CB1 receptors in sensory neurons as key determinants of cannabinoid analgesia. Our present results demonstrate that FABP5 mediates AEA transport in DRGs as its deletion elevates AEA levels, consistent with the high degree of co-expression between FABP5 and FAAH 45 . Our combined histological and behavioral data reveal that FABP5 is expressed in CB1 + sensory neurons while its ablation augments AEA levels to trigger antinociceptive effects www.nature.com/scientificreports/ mediated by CB1 receptors. The selective increase in AEA but not 2-AG levels likely suggests that monoacylglycerol lipase may maintain access to 2-AG on the plasma membrane while bypassing the need for cytosolic transport by FABP5. Alternatively, the possibility that other proteins contribute to cytosolic 2-AG trafficking cannot be excluded at this time. The molecular mechanisms underlying pain modulation by endocannabinoids have been partially elucidated with TRPV1 emerging as a major target of interest given its prominent role in pain. It was previously demonstrated that application of exogenous CB1 agonists disrupts TRPV1 sensitization in DRG neurons in vitro through a calcineurin-dependent mechanism 60,62,63 . Employing capsaicin-evoked CGRP release and NGF-mediated TRPV1 sensitization as readouts, our data revealed that FABP5 ablation elevates AEA levels and blunts TRPV1 sensitization via a CB1-and calcineurin-mediated mechanism. TRPV1 sensitization was also restored following antagonism of PPARα, consistent with a report demonstrating that PPARα physically interacts with TRPV1 and its activation desensitizes the channel 64 . Notably, these data support our previous findings that inhibition of peripherally-expressed FABP5 produces antinociceptive effects via CB1 and PPARα 45 . Unexpectedly, the inability of FAAH inhibition to block TRPV1 sensitization coupled with previous reports that FAAH inhibition promotes TRPV1 activation and pro-nociceptive effects 24,27,34-38 highlights divergent roles of FABP5 and FAAH in controlling TRPV1 function. www.nature.com/scientificreports/ There are several potential mechanisms that could underlie these contrasting effects. It is possible that FABP5 transports AEA and/or other FAAH substrates to TRPV1 for activation. Consequently, FABP5 deletion may compromise ligand delivery to TRPV1 while this function is preserved following FAAH inhibition leading to TRPV1 engagement. Additionally, it is possible that FABP5 and FAAH regulate the transport and metabolism of non-overlapping pools of TRPV1 ligands. For example, N-acyl taurines are TRPV1 agonists that are highly elevated following FAAH inhibition 26 and could in principle counteract AEA-and CB1-mediated antinociceptive effects. If N-acyl taurines are not FABP5 ligands and their levels unaltered following FABP5 inhibition, this could permit AEA-mediated antinociceptive effects to predominate. Regardless of the mechanism(s) involved, it is tempting to speculate that the inability of FAAH inhibition to temper TRPV1 sensitization may contribute at least in part to the lack of efficacy of a FAAH inhibitor in a clinical trial for pain 20 . Collectively, our results establish a key role for FABP5 in shaping endocannabinoid and N-acylethanolamine signaling in nociceptors with FABP5 emerging as a novel modulator of TRPV1 sensitization. The contrasting effects between FABP5 and FAAH coupled with previously established anti-inflammatory effects observed following FABP5 inhibition position FABP5 as a promising target for the development of analgesics 49 .

Generation of FABP5 Flox and FABP5 cKO mice.
Mice carrying LoxP-flanked exons 2 and 3 were generated via standard gene-targeting techniques by Cyagen. A 17.5 Kb targeting vector containing a diphtheria toxin (DTA) cassette, neomycin cassette flanked by Frt sites, and exons 2-3 flanked by LoxP sites was electroporated into C57Bl/6 ES cells, which were subsequently selected for blastocyst microinjections. Founders were confirmed as germline-transmitted via crossbreeding with Flp-deleter mice. The F1 progeny were subsequently backcrossed to C57Bl/6 mice and then crossed with Trpv1-Cre mice (Jackson Labs #017769) to ablate FABP5 in TRPV1 + nociceptors. Control C57Bl/6J mice were from Jackson Labs.
Hargreaves. Thermal hyperalgesia was assessed as previously reported 43 . Briefly, inflammation was induced by a unilateral, intraplantar injection of 1% carrageenan (20 µL in saline). The average latency to withdraw the paw was measured when a focused beam of radiant heat was applied to the mid plantar surface using a Hargreaves plantar apparatus (Ugo Basile). Mice were acclimated to the experimental room and Hargreaves for 2 days prior to baseline testing and at least 1 h prior to testing.
Behavioral spectrometer. Mice were acclimated to the Behavioral Sequencer box (Behavioral Instruments Inc, New Jersey) for 2 days prior to testing 51 . Mice were allowed to move around freely within the 40 cm by 40 cm arena for 30 min during testing. Using a combination of cameras, infrared sensors and accelerometers embedded in the floor, the frequency and duration of predetermined behaviors were analyzed with Viewer 3 Immunofluorescence. Mice were anesthetized with isoflurane and transcardially perfused with saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS), pH 7.4. DRGs were dissected out from the spinal column and immersed in the same fixative overnight at 4ºC. The next day the tissue was immersed in 30% sucrose in 0.1 M PB for cryoprotection. The DRGs were embedded in a gelatin-albumin mixture (3% gelatin, 30% egg albumin in dH 2 O) and frozen using a liquid nitrogen-chilled isopentane bath. Cryostat sections 16 µm in thick were thaw-mounted on gelatin/chromium-coated glass slides, air dried, and stored in a frost-free freezer at − 20 °C until further processing. For immunofluorescence staining slides were thawed at room temperature for 10 min. Sections were fixed onto the slides with 4% PFA for 3 min, rinsed with PBS three times for 10 min each, and incubated for 30 min in PBS containing 5% normal donkey serum (NDS). Slides were incubated 24-48 h at 4 °C with a mixture of primary antibodies (Table 1) diluted in PBS with 0.3% Triton X-100 and 5% NDS. Following primary antibody incubation, the sections were again washed three times for 10 min each in PBS followed by 30 min in 5% NDS in PBS. The slides were incubated for 1 h at 37 °C with a mixture of secondary antibodies conjugated to either Alexa 488 or 594 diluted in PBS with 0.3% Triton X-100 and 5% NDS. Slides were subsequently washed three times with PBS for 10 min each and immediately mounted with Vectashield Plus® antifade mounting medium with DAPI (Vector Labs Burlingame, CA, Cat# H-2000) and stored at 4 °C in the dark. Fluorescent immunoreactivity in cells was observed with a Zeiss Axioplan 2 epifluorescent microscope. Images were obtained using Zeiss AxioCam HRm monochrome digital camera, and AxioVision Rel. 4.6 microscope software. Images were only adjusted for brightness and contrast.
qPCR. RNA was extracted using the RNeasy mini kit (Qiagen) followed by cDNA synthesis using the Super-Script III First Strand synthesis system (ThermoFisher). qPCR was performed with PowerUp SYBR green (Ther-moFisher) on a StepOnePlus instrument (Applied Biosystems). Quantification was performed using the 2 −ΔΔCt method with actin serving as the housekeeping gene. The following primers were used: CGRP. CGRP release was performed as previously described 49  Dissociated DRG neurons were incubated with 5 µM fura-2AM in EC1A buffer for 30 min at 37 °C. DRG neurons loaded with fura-2AM were pulsed alternately with 340 and 380 nm wavelength illumination (Lambda XL, Sutter Instruments, Novato, CA). Images were acquired using the ORCA-Flash4.0 digital camera at a capture rate of 0.33 Hz. The fluorescence ratio for individual neurons was determined as the intensity of emission during 340 nm excitation (I340) divided by 380 nm emission (I380) and used as an indicator of change in cytoplasmic calcium. The I340/I380 ratio was calculated on a pixel-by-pixel basis using the MetaFluor software (Molecular Devices, Sunnyvale, CA, USA). NGF (2.5S murine, Gibco, Waltham, Massachusetts) at 100 ng/ml and capsaicin (Sigma-Aldrich, St Louis, MO) at 100 nM in EC1A were applied using an array of quartz fiber flow pipes (500 mm internal diameter) positioned about 1 mm away from DRGs. Drugs were added to Fura-2AM, EC1A, NGF and capsaicin solutions so that cells were continually bathed in the following concentrations: 10 µM GW6471, 1 µM AM251, 1 µM PF3845 (Cayman, Ann Arbor, MI), and 200 nM cyclosporin A (TCI, Portland, OR).
Statistical analysis. Data are represented as mean ± SD. Thermal hyperalgesia was analyzed by One-Way ANOVA followed by Tukey post-hoc while behavioral spectrometer assays were analyzed via paired t-test. Changes in lipid levels were assessed via Two-Way ANOVA followed by Bonferroni post-hoc or unpaired t-test as appropriate. Carrageenan-evoked CGRP release assays were analyzed by unpaired t-test while capsaicinevoked release was analyzed via One-Way ANOVA followed by Dunnett's post-hoc or unpaired t-test as appropriate. Calcium imaging was examined via One-Way ANOVA followed by Dunnett's post-hoc test. qPCR data were analyzed by comparing ΔCt values using an unpaired t-test.

Ethics statement. All experiments were approved by the Stony Brook University Animal Care and Use
Committee (#277150). The experiments met the United States Public Health Service Policy guidelines on Humane Care and Use of Laboratory Animals. Mice were housed in an AAALAC certified facility with ad libitum access to food and water, and lighting was maintained on a 12-h light/dark cycle. Data reporting in the manuscript follows the recommendations in the ARRIVE guideline. All experiments were performed on 8-to 12-week-old mice.

Data availability
Data will be available from the corresponding author upon reasonable request.